The present invention relates to tumor suppressor genes, in particular to xe2x80x9clatsxe2x80x9d genes (large tumor suppressor) and their encoded protein products, as well as derivatives and analogs thereof. Production of lats proteins, derivatives, and antibodies is also provided. The invention further relates to therapeutic compositions and methods of diagnosis and therapy.
Tumorigenesis in humans is a complex process involving activation of oncogenes and inactivation of tumor suppressor genes (Bishop, 1991, Cell 64:235-248). Tumor suppressor genes in humans have been identified through studies of genetic changes occurring in cancer cells (Ponder, 1990, Trends Genet. 6:213-218; Weinberg, 1991, Science 254:1138-1146). In Drosophila, tumor suppressor genes have been previously identified by recessive overproliferation mutations that cause late larval and pupal lethality (Gateff, 1978, Science 200:1448-1459; Gateff and Mechler, 1989, CRC Crit. Rev. Oncogen 1:221-245; Bryant, 1993, Trends Cell Biol. 3:31-35; Txc3x6rxc3x6k et al., 1993, Genetics 135:71-80). Mutations of interest were identified when dissection of dead larvae and pupae revealed certain overproliferated tissues. Several genes identified in homozygous mutants have been cloned including l(1)discs large-1(dlg; Woods and Bryant, 1991, cell 66:451-464; Woods and Bryant, 1993, Mechanisms of Development 44:85-89), fat (Mahoney et al., 1991, Cell 67:853-868), l(2)giant larvae (lg1. Lxc3xctzelschwab et al., 1987, EMBO J. 6:1791-1797; Jacob et al., 1987, Cell 50:215-225), expanded (ex; Boedigheimer and Laughon, 1993, Development 118:1291-1301;Boedigheimer et al., 1993, Mechanisms of Development 44:83-84), hyperplastic discs (hyd; Mansfield et al., 1994, Developmental Biology 165:507-526) and the gene encoding the S6 ribosomal protein (Watson et al., 1992, Proc. Natl. Acad. Sci. USA 89:11302-11306; Stewart and Denell, 1993, Mol. Cell. Biol. 13:2524-2535).
Although examining homozygous mutant animals has allowed the successful identification of overproliferation mutations that cause late larval and pupal lethality, mutations that cause lethality at early developmental stages are unlikely to be recovered by this approach. The present invention solves this problem by providing a method for identifying tumor suppressor genes that does not exclude genes that when mutated cause lethality in early developmental stages, and provides genes thus identified with a fundamental role in regulation of cell proliferation.
The cessation of proliferative capacity by cells in culture is termed cellular senescence. Cellular senescence is used as an experimental model for cellular aging. Normal vertebrate cells in culture have a finite lifespan in that they undergo a characteristic maximum number of population doublings. The maximum number of population doublings that a cell can undergo inversely correlates with the age of the human donor. Cells from many human tumors are immortal cell lines when grown in tissue culture, i.e., they exhibit infinite or continuous cell growth, suggesting that overcoming senescence is part of carcinogenesis. (For the foregoing see Hubbard and Ozer, 1995, xe2x80x9cSenescence and immortalization of human cells,xe2x80x9d in Cell Growth and Apoptosis, A Practical Approach, Ch. 12, Studzinski, G. P. (ed.), Oxford University Press Inc., New York, N.Y., pp. 229-248; Hubbard-Smith et al., 1992, Mol. Cell. Biol. 12:2273-2281). A comparative study of preimmortalized and immortalized human fibroblasts transformed with a defective SV40 genome has led to the suggestion that a chromosomal region at and/or distal to 6q21 plays a role in immortalization of cells (Hubbard-Smith et al., 1992, Mol. Cell. Biol. 12:2273-2281).
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
The present invention relates to nucleotide sequences of lats genes (Drosophila, human, and mouse lats and lats homologs of other species), and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. Nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences are also provided. In a specific embodiment, the lats protein is a human protein.
The invention also relates to a method of identifying tumor suppressor genes that does not exclude from identification genes that cause lethality at early developmental stages, thus overcoming the limitations of prior art methods. The method thus allows the identification of genes that regulate cell proliferation and that act at early developmental stages. The genes which thus can be identified play a fundamental role in regulation of cell proliferation such that their dysfunction (e.g., by lack of expression or mutation) leads to overproliferation and cancer.
Lats is a gene provided by the present invention, identified by the method of the invention, that acts to inhibit cell proliferation, and that plays a crucial role throughout development.
The invention also relates to lats derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) lats protein. Such functional activities include but are not limited to kinase activity, antigenicity [ability to bind (or compete with lats for binding) to an anti-lats antibody], immunogenicity (ability to generate antibody which binds to lats), and ability to bind (or compete with lats for binding) to a receptor/ligand for lats (e.g., a SH3 domain-containing protein).
The invention further relates to fragments (and derivatives and analogs thereof) of lats which comprise one or more domains of a lats protein.
Antibodies to lats, and lats derivatives and analogs, are additionally provided.
Methods of production of the lats proteins, derivatives and analogs, e.g., by recombinant means, are also provided.
The present invention also relates to therapeutic and diagnostic methods and compositions based on lats proteins and nucleic acids. Therapeutic compounds of the invention include but are not limited to lats proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the lats proteins, analogs, or derivatives; and lats antisense nucleic acids.
The invention provides for treatment of disorders of overproliferation (e.g., cancer and hyperproliferative disorders) by administering compounds that promote lats activity (e.g., lats, an agonist of lats; nucleic acids that encode lats).
The invention also provides methods of treatment of disorders involving deficient cell proliferation (growth) or in which cell proliferation is otherwise desired (e.g., degenerative disorders, growth deficiencies, lesions, physical trauma) by administering compounds that antagonize, (inhibit) lats function (e.g., antibodies, antisense nucleic acids).
In a specific embodiment, lats function is antagonized in order to inhibit cellular senescence, in vivo or in vitro.
Antagonizing lats function can also be done to grow larger animals and plants, e.g., those used as food or material sources.
Animal models, diagnostic methods and screening methods for predisposition to disorders, and methods to identify lats agonists and antagonists, are also provided by the invention.
As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in-contrast to its encoded protein product which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, xe2x80x9clatsxe2x80x9d shall mean the lats gene, whereas xe2x80x9clatsxe2x80x9d shall indicate the protein product of the lats gene.
FIG. 1. Identifying overproliferation mutations in mosaic flies. (A) Although animals that are homozygous for a lethal mutation could die at an early developmental stage, mosaic flies carrying clones of cells that are homozygous for the same mutation could live. One can identify potential tumor suppressors by generating and examining clones of overproliferated mutant cells in mosaic animals. The genetic constitution of these mosaic flies is similar to the mosaicism of the tumor patients. (B) Genetic scheme. The P-element insertions carrying the FLP recombinase (hsFLP; Golic and Lindquist, 1989, Cell 59:499-509), its target site, FRT (solid arrows, Xu and Rubin, 1993, Development 117:1223-1237), the yellow+ and mini-white+ marker genes (y+ and mini-w+, open arrows) are indicated. Mutagenized males were crossed to females to produce heterozygous embryos. Clones of cells homozygous for the induced mutations were generated in developing first-instar larvae by mitotic recombination at the FRT sites induced with the FLP recombinase. Mosaic adults were examined for overproliferated mutant patches (wxe2x88x92, yxe2x88x92). Individuals carrying clones of interest were then mated to recover the mutations of interest in the next generation (Xu and Rubin, 1993, Development 117:1223-1237; Xu and Harrison, 1994; Methods in Cell Biology 44:655-682). Clones of ommatidia derived from fast proliferating mutant cells were identified since they were larger than their darkly pigmented wt (wild-type) twin-spot clones (mini-w+/mini-w+).
FIG. 2. Mutant phenotypes. (A) A clone of unpatterned, overproliferated lats mutant cells in the eye. (B) Induced at the same stage, the 93B mutant cells formed a less overproliferated clone. (C) A third instar latse26-1 larva (right) was much larger than a wt sibling (left; at 18xc2x0 C.). (D) Wing discs from the larva in (C) (wt, top; latse26-1, bottom). (E) Dissected central nervous systems (wt, top; latse26-1, bottom). (F) A SEM (scanning electron microscope) view of a lats clone near the eye. (G) A closer view of a region in (F) showing the irregularity of the sizes and shapes of the mutant cells. (H) A plastic section of a mutant clone similar to the one in (F). Cells seem to be xe2x80x9cbuddingxe2x80x9d out of the surface to form new proliferating lobes (arrows). (I) A lats clone on the back. The boxed area is shown in (J). The bristles in the mutant clone are short, bent and often split (arrows). (K) A closer view of the hairs in a lats clone on the body showing enlarged bases and bent tips. (L) A section of a lats clone on the back showing extra cuticle deposits (arrows). All the mutant clones were induced with latsx1 unless stated differently.
FIG. 3. Organization of the Drosophila lats gene. The genomic restriction map of the lats region is aligned with the lats 5.7 kb transcript unit. The direction of transcription is indicated with large arrows. The sizes of the lats introns are as follows: intron 1 (5.0 kb), intron 2 (5.8 kb), intron 3 (68 bp), intron 4 (63 bp), intron 5 (64 bp), intron 6 (61 bp), intron 7 (62 bp). The genomic DNA from +7.5 (BglII) to xe2x88x924.2 (EcoRI) was used to screen a total imaginal disc cDNA library, which isolated three groups of cDNAs: lats, T1, T2. The introns in the T2 transcript are not labeled. Only parts of the zfh-1 (Fortini et al., 1991, Mechan. Dev. 34:113-122) and T1 transcripts are indicated. The locations of the P-element insertion (latsP1), the deletions in the five excision alleles (latse7-2, e78, e100, e119, e148) and in latsa1, latsa4 are indicated at the bottom. The slash indicates a gap in the genomic map. Restriction sites: EcoRI (small open arrow), BglII (open box) and BamHI (open circle). The BglII site at the xe2x88x920.5 position of the CLT-52 clone is not present in other genomic DNA. A scale is labeled under the restriction map.
FIG. 4. RNA blot analysis of the Drosophila lats mRNA. Five xcexcg of poly(A)+ RNA isolated from various developmental stages was separated on a 1% agarose gel, and hybridized with 32P-labeled 5xe2x80x2 end 1 kb probe from the Drosophila lats cDNA. E0-2 hrs, E2-4 hrs, E4-6 hrs, E6-8 hrs, E8-16 hrs and E16-24 hrs indicate the age of the embryos in hours. RNA from first, second and third instar larvae is denoted by L1, L2, and L3, respectively. The numbers and arrows on the right correspond to the size and location of the RNA standards. A 5.7 kb RNA was found in all the developmental stages, whereas a 4.7 kb RNA was predominantly present in 0 to 4 hour old embryos. The blot was also hybridized with DNA from the ribosomal protein gene, RNA1.
FIG. 5. Composite cDNA sequence of the Drosophila lats gene. The entire cDNA sequence (SEQ ID NO:1) corresponding to the 5.7 kb lats RNA is shown. This nucleotide sequence is a composite of two cDNA clones (nucleotide 1-191 from cDNA 9 and the rest from cDNA A2). The sequence of the corresponding genomic DNA has been determined and is identical to the cDNA sequence except where indicated (above the cDNA sequence). The predicted amino acid sequence (SEQ ID NO:2) is shown below the cDNA sequence. The opa repeat is indicated by the heavy bar. The location of the putative SH3 binding site and the RERDQ peptides are designated by dashed lines. The two sites that match the polyadenylation signal consensus sequence are underlined. The second site is located at 12 bp away from the 3xe2x80x2 end of the cDNA. The locations of the introns are indicated by vertical arrows. The underlined 141 bp sequence at the 3xe2x80x2 end of the lats transcript is identical to the 5xe2x80x2 end untranslated sequence of the class I transcript of the Drosophila phospholipase C gene, plc-21. The location of the 446 bp deletion in the lata1 allele is also indicated.
FIG. 6. Schematic of the Drosophila lats predicted protein (SEQ ID NO:2) and the related proteins (A) and sequence comparison of the proteins homologous to lats (B). In FIG. 6A, solid, hatched, open and shaded boxes denote putative SH3 binding site, opa repeat, RERDQ peptide and kinase domain in the lats protein, respectively. The Dbf20, Dbf2 and COT-1 proteins are illustrated at the bottom. The regions that are homologous to lats are indicated by shaded boxes. The degrees of sequence similarity (percentage of identical sequences inside parentheses; percentage of identical or conservative substitutions outside parentheses) between lats and the three related proteins are indicated above the corresponding regions of these proteins. In FIGS. 6B and 6C, the carboxy-terminal half of lats is compared to the six most related proteins that are revealed by blastp (a software program that searches for protein sequence homologies) search as of Sep. 1, 1994. Neurospora cot-1 (SEQ ID NO:11); tobacco PKTL7 (SEQ ID NO:12); common ice plant protein kinase (SEQ ID NO:13); spinach protein kinase (SEQ ID NO:14); yeast Dbf-20 (SEQ ID NO:15); yeast Dbf2 (SEQ ID NO:16). Amino acid residues identical to lats are highlighted. Numbers at the beginning of every sequence refer to the position of that amino acid within the total protein sequence. The boundary of the kinase domain is defined according to Hanks et al. (1988, Science 241:42-52). The location of a region of about 40 amino acid residues that is not conserved among the proteins is indicated by the heavy bar above the sequence. The sequence of PKTL7 from tobacco, Nicotiana tabacum, was submitted to Genbank by Huang,Y. (x71057). Both the sequence of the protein kinase from spinach, Spinacia oleracea, and the sequence of the protein kinase from common ice plant, Mesembryanthemum crystallinum, were submitted to Genbank by Baur, B., Winter, K., Fischer, K. and Dietz, K. (Z30329 and Z30330).
FIG. 7. cDNA sequence (SEQ ID NO:5) and deduced protein sequence (SEQ ID NO:6) of a mouse lats homolog, m-lats.
FIG. 8. cDNA sequence (SEQ ID NO:7) and deduced protein sequence (SEQ ID NO:8) of a mouse lats homolog, m-lats2.
FIG. 9. cDNA sequence (SEQ ID NO:3) and deduced protein sequence (SEQ ID NO:4) of a human lats homolog, h-lats.
FIG. 10. Schematic diagram of plasmid pBS(KS)-h-lats, containing the full length coding sequence of the h-lats cDNA.
FIG. 11. Alignment of the h-lats protein sequence (SEQ ID NO:4) (upper case letters) with the m-lats protein sequence (SEQ ID NO:6) (lower case letters). A dot indicates amino acid identity; a dash indicates a deletion relative to the sequence on the line above. The amino-terminal portion of the m-lats protein is not shown due to the missing 5xe2x80x2 end of the m-lats cDNA coding region.
FIG. 12. Alignment of the h-lats protein sequence (SEQ ID NO:4) (upper case letters) with the m-lats2 protein sequence (SEQ ID NO:8) (lower case letters). A dot indicates amino acid identity; a dash indicates a deletion relative to the sequence on the line above. The amino-terminal portion of the m-lats2 protein is not shown due to the missing 5xe2x80x2 end of the m-lats2 cDNA coding region.
FIG. 13. Alignment of the h-lats protein sequence (SEQ ID NO:4) (upper case letters) with the Drosophila lats protein sequence (SEQ ID NO:2) (lower case letters). A dot indicates amino acid identity; a dash indicates a deletion relative to the sequence on the line above. Insertions in the Drosophila sequence relative to the human sequence are indicated below the sequence line. Conserved domains are indicated. LSD2 =lats split domain 2; LSD2a=LSD2 anterior portion; LSD2p=LSD2 posterior portion. The putative SH3-binding domain and the kinase domain are shown. LSD1=lats split domain 1; LSD1a=LSD1 anterior portion; LSD1p=LSD1 posterior portion. LFD=lats flanking domain. LCD1=lats C-terminal domain 1; LCD2=lats C-terminal domain 2; LCD3=lats C-terminal domain 3.
FIG. 14. Schematic diagram of plasmid pCaSpeR-hs-h-lats, an expression vector containing the full length coding sequence of the h-lats cDNA.
FIG. 15. Northern blot analysis of h-lats expression in normal human tissues. A 32P-labeled BamHI fragment of h-lats was used as a probe for hybridization to polyA+ RNA from the normal human fetal and adult tissues indicated for each lane. The positions of standard molecular weight markers are shown at right. The positions of the h-lats RNA and of xcex2-actin RNA (used as a standard) are shown.